Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere

In recent years, outstanding molecular approaches have been used to investigate genes and functions involved in plant-microbe endosymbioses. In this review, we outline the use of proteomic analysis, based on two-dimensional electrophoresis and mass spectrometry, to characterize symbiosis-related proteins. During the last decade, proteomics succeeded in identifying about 400 proteins associated with the development and functioning of both mycorrhizal and rhizobial symbioses. Further progress in prefractionation procedures is expected to allow the detection of symbiotic proteins showing low abundance or being present in certain cell compartments.     

Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from non-mycorrhizal tomato plants

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. The more tomato plants were colonized by the arbuscular mycorrhizal fungus Glomus mosseae, the more microconidia germination was increased, indicating that alterations of the exudation pattern depended on the degree of root AM colonization. Moreover, alterations of the exudation pattern of mycorrhizal plants are not only local, but also systemic. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

The cultivation bias: different communities of arbuscular mycorrhizal fungi detected in roots from the field,from bait plants transplanted to the field,and from a greenhouse trap experiment

The community composition of arbuscular mycorrhizal fungi (AMF) was investigated in roots of four different plant species (Inula salicina, Medicago sativa, Origanum vulgare, and Bromus erectus) sampled in (1) a plant species-rich calcareous grassland, (2) a bait plant bioassay conducted directly in that grassland, and (3) a greenhouse trap experiment using soil and a transplanted whole plant from that grassland as inoculum. Roots were analyzed by AMF-specific nested polymerase chain reaction, restriction fragment length polymorphism screening, and sequence analyses of rDNA small subunit and internal transcribed spacer regions. The AMF sequences were analyzed phylogenetically and used to define monophyletic phylotypes. Overall, 16 phylotypes from several lineages of AMF were detected. The community composition was strongly influenced by the experimental approach, with additional influence of cultivation duration, substrate, and host plant species in some experiments. Some fungal phylotypes, e.g., GLOM-A3 (Glomus mosseae) and several members of Glomus group B, appeared predominantly in the greenhouse experiment or in bait plants. Thus, these phylotypes can be considered r strategists, rapidly colonizing uncolonized ruderal habitats in early successional stages of the fungal community. In the greenhouse experiment, for instance, G. mosseae was abundant after 3 months, but could not be detected anymore after 10 months. In contrast, other phylotypes as GLOM-A17 (G. badium) and GLOM-A16 were detected almost exclusively in roots sampled from plants naturally growing in the grassland or from bait plants exposed in the field, indicating that they preferentially occur in late successional stages of fungal communities and thus represent the K strategy. The only phylotype found with high frequency in all three experimental approaches was GLOM A-1 (G. intraradices), which is known to be a generalist. These results indicate that, in greenhouse trap experiments, it is difficult to establish a root-colonizing AMF community reflecting the diversity of these fungi in the field roots because fungal succession in such artificial systems may bias the results. However, the field bait plant approach might be a convenient way to study the influence of different environmental factors on AMF community composition directly under the field conditions. For a better understanding of the dynamics of AMF communities, it will be necessary to classify AMF phylotypes and species according to their life history strategies.     

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.     

Responses of fungal root colonization,plant cover and leaf nutrients to long‐term exposure to elevated atmospheric CO2 and warming in a subarctic birch forest understory

Responses of the mycorrhizal fungal community in terrestrial ecosystems to global change factors are not well understood. However, virtually all land plants form symbiotic associations with mycorrhizal fungi, with approximately 20% of the plants' net primary production transported down to the fungal symbionts. In this study, we investigated how ericoid mycorrhiza (ErM), fine endophytes (FE) and dark septate endophytes (DSE) in roots responded to elevated atmospheric CO₂ concentrations and warming in the dwarf shrub understory of a birch forest in the subarctic region of northern Sweden. To place the belowground results into an ecosystem context we also investigated how plant cover and nutrient concentrations in leaves responded to elevated atmospheric CO₂ concentrations and warming. The ErM colonization in ericaceous dwarf shrubs increased under elevated atmospheric CO₂ concentrations, but did not respond to warming following 6 years of treatment. This suggests that the higher ErM colonization under elevated CO₂ might be due to increased transport of carbon belowground to acquire limiting resources such as N, which was diluted in leaves of ericaceous plants under enhanced CO₂. The elevated CO₂ did not affect total plant cover but the plant cover was increased under warming, which might be due to increased N availability in soil. FE colonization in grass roots decreased under enhanced CO₂ and under warming, which might be due to increased root growth, to which the FE fungi could not keep up, resulting in proportionally lower colonization. However, no responses in aboveground cover of Deschampsia flexuosa were seen. DSE hyphal colonization in grass roots significantly increased under warmer conditions, but did not respond to elevated CO₂. This complex set of responses by mycorrhizal and other root‐associated fungi to global change factors of all the fungal types studied could have broad implications for plant community structure and biogeochemistry of subarctic ecosystems.     

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a <Emphasis Type="Italic">Trichoplusia ni</Emphasis> cell line

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0–15 μmol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 μmol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.     

Cophenetic correlation analysis as a strategy to select phylogenetically informative proteins: an example from the fungal kingdom

Background  

The construction of robust and well resolved phylogenetic trees is important for our understanding of many, if not all biological processes, including speciation and origin of higher taxa, genome evolution, metabolic diversification, multicellularity, origin of life styles, pathogenicity and so on. Many older phylogenies were not well supported due to insufficient phylogenetic signal present in the single or few genes used in phylogenetic reconstructions. Importantly, single gene phylogenies were not always found to be congruent. The phylogenetic signal may, therefore, be increased by enlarging the number of genes included in phylogenetic studies. Unfortunately, concatenation of many genes does not take into consideration the evolutionary history of each individual gene. Here, we describe an approach to select informative phylogenetic proteins to be used in the Tree of Life (TOL) and barcoding projects by comparing the cophenetic correlation coefficients (CCC) among individual protein distance matrices of proteins, using the fungi as an example. The method demonstrated that the quality and number of concatenated proteins is important for a reliable estimation of TOL. Approximately 40–45 concatenated proteins seem needed to resolve fungal TOL.     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Similarity and diversity in adventitious root anatomy as related to root aeration among a range of wetland and dryland grass species

Assessments of the anatomy, porosity and profiles of radial O₂ loss from adventitious roots of 10 species in the Poaceae (from four subfamilies) and two species in the Cyperaceae identified a combination of features characteristic of species that inhabit wetland environments. These include a strong barrier to radial O₂ loss in the basal regions of the adventitious roots and extensive aerenchyma formation when grown not only in stagnant but also in aerated nutrient solution. Adventitious root porosity was greater for plants grown in stagnant compared with aerated solution, for all 10 species in the Poaceae. The ‘wetland root’ archetype was best developed in Oryza sativa and the two species of the Cyperaceae, in which the stele contributed less than 5% of the root cross‐sectional area, the cells of the inner cortex were packed in a cuboidal arrangement, and aerenchyma was up to 35–52%. Variations of this root structure, in which the proportional and absolute area of stele was greater, with hexagonal arrangements of cells in the inner cortex and varying in the extent of aerenchyma formation, were present in the other wetland species from the subfamilies Pooideae, Panicoideae and Arundinoideae. Of particular interest were Vetiveria zizanoides and V. filipes, wetland grass species from the tribe Andropogoneae (the same tribe as sorghum, maize and sugarcane), that had a variant of the root anatomy found in rice. The results are promising with regard to enhancing these traits in waterlogging intolerant crops.     

Common anastomosis and internal transcribed spacer RFLP groupings in binucleate Rhizoctonia isolates representing root endophytes of Pinus sylvestris, Ceratorhiza spp. from orchid mycorrhizas and a phytopathogenic anastomosis group

Binucleate Rhizoctonia endophytes from the roots of nursery-grown Pinus sylvestris (Scots pine) seedlings and the orchid Goodyera repens from Scots pine forests were characterized on the basis of morphological characters, anastomosis group membership and PCR-assisted ribosomal DNA fingerprinting. Common hyphal and colony morphological traits displayed by the Finnish binucleate Rhizoctonia isolates and a range of Canadian orchid root endophytes enabled them to be placed in the anamorphic genus Ceratorhiza . Five main anastomosis groups were identified and included groups that contained different combinations of Scots pine, G. repens and Canadian Ceratorhiza spp. isolates. Two Scots pine root endophytes anastomosed with a phytopathogenic Japanese tester isolate, confirming their membership of anastomosis group I, which is known to include the teleomorphic species Ceratobasidium cornigerum . Hierarchical cluster analysis of RFLPs in the internal transcribed spacer of ribosomal DNA enabled the division of isolates into one of five RFLP groups. The RFLP and anastomosis groupings were closely correlated; isolates within each of four RFLP groups, which shared 100% RFLP identity, anastomosed in the cross-pairing anastomosis group tests. However, all represented different vegetatively compatible populations (clones) because the diagnostic killing reaction, a cellular vegetative incompatibility response, was identified at hyphal fusion junctions. These findings indicate a high degree of intraspecific variation within Ceratobasidium cornigerum , which includes isolates able to enter into either mutualistic or pathogenic root association with susceptible host plants. The common anastomosis group/RFLP groupings identified also strongly support the hypothesis that conifer tree roots can act as large inoculum reservoirs for these orchid endophytes, allowing the development of inter-plant connections, via commonly shared hyphal linkages, in boreal forest ecosystems.     

Analysis of root images from auger sampling with a fast procedure: a case of application to sugar beet

Manual line-intersect methods for estimating root length are being progressively replaced by faster and more accurate image analysis procedures. These methods even allow the estimation of some more root parameters (e.g., diameter), but still require preliminary labour-intensive operations. Through a task-specific macro function written in a general-purpose image analysis programme (KS 300 – Zeiss), the processing time of root images was greatly reduced with respect to skeletonisation methods by using a high-precision algorithm (Fibrelength). This has been previously proposed by other authors, and estimates length as a function of perimeter and area of the digital image of roots. One-bit binary images were acquired, aiming at large savings in computer memory, and automatic discrimination of roots against extraneous objects based on their elongation index (perimeter²/area), was performed successfully. Of four tested spatial resolutions (2.9, 5.9, 8.8, 11.8 pixel mm^–1), in clean samples good accuracy in root length estimation was achieved at 11.8 pixel mm^–1, up to a root density of 5 cm cm^–2 on the scanner bed. This resolution is theoretically suitable for representing roots at least 85 m wide. When dealing with uncleaned samples, a thick layer of water was useful in speeding up spreading of roots on the scanner bed and avoiding underestimation of their length due to overlaps with organic debris. A set of fibrous root samples of sugar beet (Beta vulgaris var. saccharifera L.) collected at harvest over two years at Legnaro (NE Italy) was analysed by applying the above procedure. Fertilisation with 100 kg ha^–1 of nitrogen led to higher RLD (root length density in soil) in shallow layers with respect to unfertilised controls, whereas thicker roots were found deeper than 80 cm of soil without nitrogen.     

Alien plants associate with widespread generalist arbuscular mycorrhizal fungal taxa: evidence from a continental‐scale study using massively parallel 454 sequencing

Aim The biogeography of arbuscular mycorrhizal (AM) fungi is poorly understood, and consequently the potential of AM fungi to determine plant distribution has been largely overlooked. We aimed to describe AM fungal communities associating with a single host‐plant species across a wide geographical area, including the plant’s native, invasive and experimentally introduced ranges. We hypothesized that an alien AM plant associates primarily with the geographically widespread generalist AM fungal taxa present in a novel range. Location Europe, China. Methods We transplanted the palm Trachycarpus fortunei into nine European sites where it does not occur as a native species, into one site where it is naturalized (Switzerland), and into one glasshouse site. We harvested plant roots after two seasons. In addition, we sampled palms at three sites in the plant’s native range (China). Roots were subjected to DNA extraction, polymerase chain reaction (PCR) and 454 sequencing of AM fungal sequences. We analysed fungal communities with non‐metric multidimensional scaling (NMDS) ordination and cluster analysis and studied the frequency of geographically widespread fungal taxa with log‐linear analysis. We compared fungal communities in the roots of the palm with those in resident plants at one site in the introduced range (Estonia) where natural AM fungal communities had previously been studied. Results We recorded a total of 73 AM fungal taxa. AM fungal communities in the native and introduced ranges differed from one another, while those in the invasive range contained taxa present in both other ranges. Geographically widespread AM fungal taxa were over‐represented in palm roots in all regions, but especially in the introduced range. At the Estonian site, the palm was colonized by the same community of widespread AM fungal taxa as associate with resident habitat‐generalist plants; by contrast, resident forest‐specialist plants were colonized by a diverse community of widespread and other AM fungal taxa. Main conclusions AM fungal communities in the native, invasive and experimentally introduced ranges varied in taxonomic composition and richness, but they shared a pool of geographically widespread, non‐host‐specific taxa that might support the invasion of a generalist alien plant. Our dataset provides the first geographical overview of AM taxon distributions obtained using a single host‐plant species.     

SMAD‐proteins as a molecular switch from hypertrophy to apoptosis induction in adult ventricular cardiomyocytes

Heart failure development goes along with a transition from hypertrophic growth to apoptosis induction. In adult cardiomyocytes SMAD proteins are only activated under apoptotic, but not under hypertrophic conditions and are increased at the transition to heart failure. Therefore, SMADs could be candidates that turn the balance from hypertrophic growth to apoptosis resulting in heart failure development. To test this hypothesis we infected isolated rat ventricular cardiomyocytes with adenovirus encoding SMAD4 (AdSMAD4) and investigated the impact of SMAD4 overexpression on the development of apoptosis and hypertrophy under stimulation with phenylephrine (PE). Infection of cardiomyocytes with AdSMAD4 significantly enhanced SMAD‐binding activity while apoptosis after 24 and 36 h infection did not rise. But when SMAD4 overexpressing cardiomyocytes were incubated with PE (10 µM), the number of apoptotic cells increased (Ctrl: 94.97 ± 6.91%; PE: 102.48 ± 4.78% vs. AdSMAD4 + PE: 118.64 ± 3.28%). Furthermore expression of caspase 3 as well as bax/bcl2 ratio increased in SMAD4 overexpressing, PE‐stimulated cardiomyocytes. In addition, the effects of SMAD4 overexpression on PE‐induced hypertrophic growth were analyzed. Protein synthesis 36 h after AdSMAD4 infection was comparable to control cells, whereas the increase in protein synthesis stimulated by phyenylephrine was significantly reduced in SMAD4 overexpressing cells (134.28 ± 10.02% vs. 100.57 ± 8.86%). SMAD4 triggers the transition from hypertrophy to apoptosis in ventricular cardiomyocytes. Since SMADs are increased under several pathophysiological conditions in the heart, it can be assumed that it triggers apoptosis induction and therefore contributes to negative remodeling and heart failure progression. J. Cell. Physiol. 220: 515–523, 2009. © 2009 Wiley‐Liss, Inc.     

Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.